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Thursday, 12 December 2013

CRISPRs - Clustered Regularly Interspaced Short Palindromic Repeats

Principles of Molecular Virology, Chapter 6 (Virus Infection), discusses immune responses to virus infection. Around ten years ago we realised that the natural world is a soup of bacteriophages. So how do bacteria survive against this constant onslaught?

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short, direct repeats of DNA base sequences. Each CRISPR contains a series of bases followed by the same series in reverse (a palindrome) and then by 30 or so base pairs known as "spacer" DNA. The spacers are short segments of virus or plasmid DNA. CRISPRs are found in the genomes of approximately 50% of bacteria and 90% of archaea. CRISPR loci are typically located on the bacterial chromosome, although some are found on plasmids. Bacteria may contain more than one CRISPR locus - up to 18 in some cases.

CRISPRs function as a sort of prokaryotic immune system, conferring resistance to exogenous genetic elements such as plasmids and bacteriophages. Intriguingly, the CRISPR system provides a form of acquired immunity, allowing the cell to remember and respond to sequences it has encountered before.

How do CRISPRs work?
CRISPRs are often adjacent to cas (CRISPR-associated) genes. The cas genes encode a large and heterogeneous family of proteins including nucleases, helicases, polymerases and polynucleotide-binding proteins, forming the CRISPR/Cas system. (Note: cas = genes, Cas = the proteins encoded by these genes.) The interesting bits are the unique spacer elements (derived from exogenous sequences such as viruses and plasmids) rather than the repeats themselves. The spacer elements originate from exogenous DNA the bacterium (or its ancestors) has previous encountered - they are typically pieces of phage or plasmid DNA. This allows the cell to recognise these sequences via base homology if they enter the cell again, e.g. if the bacterium is infected with a bacteriophage whose genome contains this sequence:

cas-encoded nucleases cleave invading DNA into short pieces.

Other cas proteins allow a fragment of the foreign DNA to be incorporated as a novel repeat-spacer unit at the leader end of the CRISPR site.

The CRISPR array is then transcribed to form a pre-CRISPR RNA (crRNA) transcript.

The pre-crRNA is cleaved within the repeat sequence by Cas proteins to generate small CRISPR RNAs, crRNAs.

The crRNAs to work in a similar way to RNAi in eukaryotic cells, although there are important differences in the machinery by which this happens.

The phages fight back
CRISPRs are an important way in which bacteria are able to survive constant attack by bacteriophages in the environment, but phages have been around for a very long time too, so they must have found ways of counteracting the CRISPR system. Eukaryotic viruses may express inhibitors such as dsRNA-binding proteins that interfere with the RNA silencing machinery, whereas bacteriophages acquire mutations or recombine the sequence corresponding to the CRISPR spacer to avoid recognition in an analogous way to how viruses of eukaryotes acquire mutations in B cell and T cell epitopes in proteins to evade the mammalian immune system.

So who (apart from bacteria) cares about CRISPRs?
Altering the spacer via genetic manipulation can provide novel phage resistance, whereas spacer deletion result in loss of phage resistance. Although CRISPRs originate in bacteria, they also work in eukaryotic cells if introduced by genetic engineering. This provides a convenient way of targeting genes in cells, including human cells. Recent work suggests that CRISPRs might also be involved in control of bacterial gene expression as well as in immunity. We will undoubtedly see much more widespread use of CRISPRs in biotechnology over the next few years, but important biological questions remain unanswered. CRISPR systems have been detected in around 50% of bacterial species so far - but what about the 50% of species which appear to lack them? How do they survive in the bacteriophage soup of the environment?